Description

Lactose (C12H22O11), also called milk sugar, is a disaccharide that consists of β-D-galactose and α/β-D-glucose through a β1-4 glycosidic linkage. Lactose is the major sugar and makes up 2–8% of milk. Simple, direct and high-throughput assays for lactose determination find wide applications. BioAssay Systems assay uses specific enzyme-coupled reactions in which lactose is cleaved and the resulting galactose forms a colored product. The color intensity at 570nm or fluorescence intensity at 530nm/585nm is directly proportional to the lactose concentration in the sample.

Key features

Use as little as 20 mL samples. Linear detection range in 96-well plate: 17 to 2000 mM lactose for colorimetric assays and 6 to 100 mM for fluorimetric assays.

Applications:

Assays of lactose in milk and other biological samples.

Drug Discovery/Pharmacology: effects of drugs on lactose metabolism.

Food and Beverages: lactose in food and beverages products.

Kit Contents:

  Assay Buffer:  10 mL    Enzyme Mix: 120 mL      Lactase:  120 mL      

  Dye Reagent: 120 mL    Standard: 1 mL 20 mM Lactose

Storage conditions. The kit is shipped on dry ice. Store all components at -20°C. Shelf life of three months after receipt.

Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.

Coloriemtric Procedure

Note: (1) glycerol and SH-containing reagents (e.g. b–mercaptoethanol, dithiothreitol) are known to interfere in this assay and should be avoided in sample preparation. (2) For samples containing galactose, a sample blank is necessary (see Procedure); (3) This assay is based on a kinetic reaction. To ensure identical incubation time, addition of Working Reagent to standard and samples should be quick and mixing should be brief but thorough. Use of a multi-channel pipettor is recommended.

Sample treatment: Milk samples should be cleared by mixing 600 mL milk with 100 mL 6 N HCl. Centrifuge 5 min at 14,000 rpm. Transfer 300 mL supernatant into a clean tube and neutralize with 50 mL 6 N NaOH. The neutralized supernatant is ready for assay (dilution factor n = 1.36).

1. Equilibrate all components to room temperature. During experiment, keep thawed Lactase and Enzyme Mix in a refrigerator or on ice.

2. Standards and samples: prepare 400 mL 2000 mM Standard by mixing 40 mL 20 mM standard with 360 mL dH₂O. Dilute standard in dH₂O as follows.

Transfer 20 mL standards and 20 mL samples into separate wells of a clear flat-bottom 96-well plate. Note: if a sample is known to contain galactose, transfer 20 mL sample in duplicate (one sample and one sample blank).

3. Reaction. For each reaction well, mix 85 mL Assay Buffer, 1 mL Lactase, 1 mL Enzyme Mix (vortex briefly before pipetting), and 1 mL Dye Reagent in a clean tube. (Note: for the sample blanks, prepare a control Working Reagent  which is the same except  WITHOUT the 1 mL Lactase).

Transfer 80 mL Working Reagent into each reaction (and control) well. Tap plate to mix. Incubate 30 min at room temperature.

4. Read optical density at 570nm (550-585nm).

Fluorimetric Procedure

For fluorimetric assays, the linear detection range is 6 to 100 mM lactose. Prepare 100 mM lactose standard by mixing 5 mL 20 mM standard with 995 mL H2O. Then dilute standards in H2O (see Colorimetric Procedure) to 100, 80, 60, 40, 30, 20, 10 and 0 mM.

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